ANF is released into the circulation in response to volume expansion, and elicits numerous physiological effects which act in a combinatorial fashion to reduce blood pressure. The physiological responses of ANF include vasodilation, natriuresis, diuresis, inhibition of the renin-angiotensin- aldosterone axis, inhibition of AVP secretion and modulation of neural activity. While the effects of acute ANF administration are well characterized, the chronic role of the hormone in cardiovascular homeostasis has remained controversial. In an effort to directly address this issue, we have generated a transgenic model system which exhibits chronically elevated levels of ANF in the systemic circulation. These transgenic animals, designated TTR-ANF, have immuno-reactive plasma ANF levels which are approximately 10 fold elevated as compared to their non- transgenic litter mates. Moreover, the TTR-ANF mice have a mean arterial blood pressure (as determined in conscious resting mice with indwelling annulus) of 75.5 plus/minus 9.9 mm Hg as compared to 103.9 plus/minus 2.0 for the non-transgenic litter mates. This study clearly demonstrates that chronically elevated levels of ANF can induce a sustained hypotensive response. In this proposal, we will continue to expand on our transgenic model. Specifically, we will: (1) Further assess the physiological consequences of elevated ANF levels in the TTR-ANF transgenic mice. These studies will include a detailed analysis of kidney function (nephron puncture and micro-catheterization analyses), assessment of baroreceptor gain, characterization of the response of the various ANF receptors, and assessment of the resistance of the transgenic animals to experimentally induced hypertension. (2) Generate transgenic mice in which an "activated" ANF receptor is expressed in the cell types which are responsive to the hormone. Studies by other groups have demonstrated that deletion of the kinsae domain in the ANF-A receptor results in constitutive guanylate cyclase activity. Expression of such a "trans-dominant" ANF receptor can be targeted to specific cell types which are responsive to ANF. This approach will allow us to experimentally establish correlates between specific target tissue activation and physiological response in intact animals. (3) Generate transgenic models with chronically elevated ANF levels in the central nervous system. Numerous studies have shown that ANF cannot breach the blood brain barrier, and that intra-cerebral injection of ANF can elicit discrete physiological responses. We will generate a transgenic model system in which ANF is specifically secreted into the cerebrospinal fluid in order to assess the consequences of chronically elevated hormone levels in the CNS. (4) Generate transgenic animals which do not synthesize ANF. Recent advances in mouse embryology have made it possible to genetically inactivate specific genes in embryonic stem (ES) cells. These cells retain a certain degree of pluripotency, and have been used to generate chimeric animals in which cells that carry the mutated allele have populated the germline. These chimeric animals have subsequently passed the mutation on to progeny mice. We will use this approach to generate animals which fail to express ANF. All of the transgenic models will be subjected to comprehensive molecular and physiological analyses to assess the consequences or altered ANF expression (or the consequences of altered activity of the ANF signal transduction pathway). These experiments will enable us to understand the physiological role that ANF exerts in chronic cardiovascular regulation.